RESUMO
The present report further analyzes the survival promoting effect of high potassium, a condition that mimics neural activity in cultured cerebellar granule cells, an excellent model to study trophic mechanisms induced by depolarization and trophic factors. We found that the survival promoting effect measured at 7 days in vitro (DIV 7) of depolarizing potassium concentrations (25 mM KCl), added at DIV2, is partially prevented by adding at DIV 2 the non-competitive NMDA blocker MK801 (10 microM). The concentration of MK801 used blocks completely the survival promoting effect of a supramaximal effective concentration of NMDA (100 microM). The addition at DIV 2 of anti-brain derived neurotrophic factor (anti-BDNF) antibody, failed to modify the effect of high potassium. The present report provides evidences that in cultured cerebellar granule cells, high potassium-induced survival promoting effect is due in part by the activation of NMDA receptors. The effect does not require the presence of BDNF.
Assuntos
Córtex Cerebelar/metabolismo , Neurônios/metabolismo , Potássio/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos/farmacologia , Fator Neurotrófico Derivado do Encéfalo/antagonistas & inibidores , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Córtex Cerebelar/efeitos dos fármacos , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
An increasing body of evidence supports a trophic action of gamma-aminobutyric acid (GABA) during nervous system development. The purported mediator of these trophic effects is a depolarizing response triggered by GABA, which elicits a calcium influx in immature CNS cells. This Mini-Review focuses on the neurotrophic role of neural activity and GABA and some of the most common intracellular cascades activated by depolarization and trophic factors. Several biological effects induced by GABA in the developing nervous system are reviewed, with particular emphasis on what is known about calcium-dependent neurotrophic effects induced by GABA and its intracellular mechanisms.
Assuntos
Cálcio/fisiologia , Movimento Celular/efeitos dos fármacos , Fosfotransferases/fisiologia , Ácido gama-Aminobutírico/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Diferenciação Sexual/fisiologiaRESUMO
During neuronal development, GABAA-mediated responses are depolarizing and induce an increase in the intracellular calcium concentration. Since calcium oscillations can modulate neurite outgrowth, we explored the capability of GABA to induce changes in cerebellar granule cell morphology. We find that treatment with GABA (1-1000 microm) induces an increase in the intracellular calcium concentration through the activation of GABA(A) receptors and voltage-gated calcium channels of the L-subtype. Perforated patch-clamp recordings reveal that this depolarizing response is due to a chloride reversal potential close to - 35 mV. When cells are grown in depolarizing potassium chloride concentrations, a shift in reversal potential (Erev) for GABA is observed, and only 20% of the cells are depolarized by the neurotransmitter at day 5 in vitro. On the contrary, cells grown under resting conditions are depolarized after GABA application even at day 8. GABA increases the complexity of the dendritic arbors of cerebellar granule neurons via a calcium-dependent mechanism triggered by voltage-gated calcium channel activation. Specific blockers of calcium-calmodulin kinase II and mitogen-activated protein kinase kinase (KN93 and PD098059) implicate these kinases in the intracellular pathways involved in the neuritogenic effect of GABA. These data demonstrate that GABA exerts a stimulatory role on cerebellar granule cell neuritogenesis through calcium influx and activation of calcium-dependent kinases.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/farmacologia , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Células Cultivadas , Cerebelo/citologia , Inibidores Enzimáticos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
Different mitochondrial nitric-oxide synthase (mtNOS) isoforms have been described in rat and mouse tissues, such as liver, thymus, skeletal muscle, and more recently, heart and brain. The modulation of these variants by thyroid status, hypoxia, or gene deficiency opens a broad spectrum of mtNOS-dependent tissue-specific functions. In this study, a new NOS variant is described in rat brain with an M(r) of 144 kDa and mainly localized in the inner mitochondrial membrane. During rat brain maturation, the expression and activity of mtNOS were maximal at the late embryonic stages and early postnatal days followed by a decreased expression in the adult stage (100 +/- 9 versus 19 +/- 2 pmol of [(3)H]citrulline/min/mg of protein, respectively). This temporal pattern was opposite to that of the cytosolic 157-kDa nNOS protein. Mitochondrial redox changes followed the variations in mtNOS activity: mtNOS-dependent production of hydrogen peroxide was maximal in newborns and decreased markedly in the adult stage, thus reflecting the production and utilization of mitochondrial matrix nitric oxide. Moreover, the activity of brain Mn-superoxide dismutase followed a developmental pattern similar to that of mtNOS. Cerebellar granular cells isolated from newborn rats and with high mtNOS activity exhibited maximal proliferation rates, which were decreased by modifying the levels of either hydrogen peroxide or nitric oxide. Altogether, these findings support the notion that a coordinated modulation of mtNOS and Mn-superoxide dismutase contributes to establish the rat brain redox status and participate in the normal physiology of brain development.
Assuntos
Encéfalo/crescimento & desenvolvimento , Mitocôndrias/enzimologia , Óxido Nítrico Sintase/metabolismo , Envelhecimento , Animais , Encéfalo/enzimologia , Cálcio/farmacologia , Fracionamento Celular , Desenvolvimento Embrionário e Fetal , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Variação Genética , Cinética , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Ratos , Ratos Wistar , Partículas Submitocôndricas/enzimologiaRESUMO
In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+ ]e under serum-free conditions. We found that 25 mm KCl (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Cerebelo/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Cerebelo/citologia , Meios de Cultura Livres de Soro/farmacologia , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , MAP Quinase Quinase 1 , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuritos/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
Se investigaron los efectos de agonistas y antagonistas alfa -adrenérgicos y dopaminérgicos sobre los receptores presinápticos de la porción prostática del conducto defrerente de rata. La variable estudiada fue el primer componente (250 ms) de la respuesta motora inducida por estimulación eléctrica de campo (pulso único). Con el objeto de bloquear los sitios de pérdida de las aminas, todos los experimentos se llevaron a cabo en presencia de cocaína 30 micronmol/l e hidrocortisona 28 micronmol/l. Asimismo, se empleó 1-propranolol 0.3 micronmol/l para bloquear los receptores ß -adrenérgiocs. Clonidina, noradrenalina (NA) y dopamina (DA) inhibieron la respuesta motora inducida por la aplicación de un pulso eléctrico. Este efecto fue, para los tres agonistas, dependiente de las concentraciones utilizadas. DA fue 10 y 10**4 veces menos potente que NA y clonidina respectivamente. El agonista selectivo D2, LY 141865, no logró inhibir la respuesta motora inclusive a una alta concentración (30 micronmol/l). Yohimbina (0.1, 0.3 y 1 micronmol/l) antagonizó en forma competitiva el efecto inhibitorio de clonidina, NA y DA, presentando valores similares de -log KB (7.57, 7.68 y 7.09 respectivamente). De manera análoga, idaxozán 0.03 micronmol/l bloqueó el efecto inhibitorio de DA con una potencia similar (- log KB = 7.81) a la de yohimbina. Por otra parte, pimozide 0.21 micronmol/l y Schering 23390 3 micronmol/l antagonizaron el efecto inhibitorio de DA, mostrando una potencia menor que los antagonistas alfa2 -adrenérgicos...
Assuntos
Ratos , Animais , Masculino , Clonidina/farmacologia , Dopamina/farmacologia , Neurônios Motores/efeitos dos fármacos , Receptores de Neurotransmissores/efeitos dos fármacos , Sódio/farmacologia , Ducto Deferente/efeitos dos fármacos , Estimulação Elétrica , Condução Nervosa/efeitos dos fármacosRESUMO
Se investigaron los efectos de agonistas y antagonistas alfa -adrenérgicos y dopaminérgicos sobre los receptores presinápticos de la porción prostática del conducto defrerente de rata. La variable estudiada fue el primer componente (250 ms) de la respuesta motora inducida por estimulación eléctrica de campo (pulso único). Con el objeto de bloquear los sitios de pérdida de las aminas, todos los experimentos se llevaron a cabo en presencia de cocaína 30 micronmol/l e hidrocortisona 28 micronmol/l. Asimismo, se empleó 1-propranolol 0.3 micronmol/l para bloquear los receptores ß -adrenérgiocs. Clonidina, noradrenalina (NA) y dopamina (DA) inhibieron la respuesta motora inducida por la aplicación de un pulso eléctrico. Este efecto fue, para los tres agonistas, dependiente de las concentraciones utilizadas. DA fue 10 y 10**4 veces menos potente que NA y clonidina respectivamente. El agonista selectivo D2, LY 141865, no logró inhibir la respuesta motora inclusive a una alta concentración (30 micronmol/l). Yohimbina (0.1, 0.3 y 1 micronmol/l) antagonizó en forma competitiva el efecto inhibitorio de clonidina, NA y DA, presentando valores similares de -log KB (7.57, 7.68 y 7.09 respectivamente). De manera análoga, idaxozán 0.03 micronmol/l bloqueó el efecto inhibitorio de DA con una potencia similar (- log KB = 7.81) a la de yohimbina. Por otra parte, pimozide 0.21 micronmol/l y Schering 23390 3 micronmol/l antagonizaron el efecto inhibitorio de DA, mostrando una potencia menor que los antagonistas alfa2 -adrenérgicos... (AU)